Test kit for determining the level of ascorbic acid in a patient&#39;s body

ABSTRACT

A method of administration of vitamin C to ensure that a continuously-saturated level is produced in the body of a taker. A dose of about 500 mg taken approximately every 12 hours produces a continuously-detectable level of vitamin C in the urine of an average healthy person, which corresponds to a sufficiently high pool of ascorbic acid in the body to provide anti-oxidant protection. The minimum dosage and regimen found to be effective are respectively substantially higher than the U.S. recommended daily allowance and more frequent than administration rates previously used in clinical trials. Also claimed are kits that permit individuals to monitor for elevated urinary excretion of useful substances which are water soluble, excreted in urine, and non-toxic at physiologically beneficial levels, such that optimal dosages and regimens can be determined.

RELATED APPLICATION

This application is a Divisional Application of U.S. patent applicationSer. No. 08/317,311, filed Oct. 3, 1994, now U.S. Pat. No. 5,558,870.

FIELD OF THE INVENTION

Vitamin C, ascorbic acid, has long been known to prevent scurvy and morerecently has been shown to have an effect on the healing of wounds, thehealth of gums, and the strength of bones through the stabilization ofcollagen. See Davies, M. B. et al., "Vitamin C: Its Chemistry andBiochemistry," Cambridge, Royal Society of Chemistry, 1991, pp. 7-25;Stare, F. J. and I. M. Stare, "Charles Glen King, 1896-1988," J. Nutr.,118:1272-7, 1988; and Roig, M. G. et al., "L-Ascorbic Acid: anOverview," Int. J. Food Sci. Nutr., 44:59-72, 1993. However, otherpossible health benefits, including prevention of cancer (see Roig,supra; Block G., "Vitamin C, Cancer and Aging," Age, 16:55-8, 1993;Marwick, C., "Cancer institute Takes a Look at Ascorbic Acid," JAMA,264:1926, 1990; and Wittes, R. E., "Vitamin C and Cancer" New Engl. J.Med., 312:178-9, 1985), prevention of heart attacks and reduction ofcholesterol (Burr, M. L. et al., "Incidence for Premature Rupture ofMembranes in Pregnant Women with Low Leukocyte Levels of Vitamin C,"Eur. J. Clin. Nutr., 39c:387-8, 1985; Kimura, H. et al., "DietaryAscorbic Acid Depresses Plasma and Low Density Lipoprotein LipidPeroxidation in Genetically Scorbutic Rats," J. Nutr., 122:1904-9, 1992;and Uchida, K. et al., "Effect of Vitamin C Depletion on SerumCholesterol and Lipoprotein Levels in ODS (od/od) Rats Unable toSynthesize Ascorbic Acid," J. Nutr., 120:1140-7, 1990), and as a boostto the immune system to prevent colds (Blanchard, J. et al. "Comparisonof Plasma, Mononuclear and Polymorphonuclear Leukocyte Vitamin C Levelsin Young and Elderly Women during Depletion and Supplementation," Eur.J. Clin. Nutr., 43:97-106, 1989; Chavance, M. et al. "Vitamin Status,Immunity and Infections in an Elderly Population," Eur. J. Clin. Nutr.,43:827-35, 1989; Vallance, S., "Platelets, Leukocytes and Buffy LayerVitamin C After Surgery," Hum. Nutr., 40c:35-41, 1986: and Vojdani, A.and M. Ghoneum, "In Vivo Effect of Ascorbic Acid Enhancement of HumanNatural Killer Cell Activity," Nutr. Res., 13:753, 1993), remaincontroversial. The "free radical theory of aging" has been postulated toexplain age-related cell damage in animals and plants. See Pryor, W. A.,"The Formation of Free Radicals and the Consequences of their Reactionsin Vivo, " Photochem. Photobiol., 28:787-801, 1978; and Harman, D., "TheAging Process," Proc. Natl. Acad. Sci. USA, 78:7124-7128, 1981. VitaminC appears to play a synergistic role with vitamin E in providingessential anti-oxidant protection (Tappel, A. L., "Vitamin E as theBiological Lipid Antioxidant" Vitam. Horm., 20:493-510, 1962; and NikiE. et al., "Inhibition of Oxidation of Methyl-Linoleate in Solution byVitamin E and Vitamin C," J. Biol. Chem., 259:4177-4182, 1984). Giventhe large number of "free radical" diseases, it is reasonable to assumethat taking an optimal dose of vitamin C could be extremely beneficial,as suggested by Block, G., supra, and by Harman, D. in "Free RadicalTheory of Aging: Current Status" Lipofuscin 1987: State of the Art,edited by I. Zs.-Nagy, New York, Elsevier, 1988, pp. 3-21. With around20 million people in the U.S. taking daily supplements of Vitamin C, itis appropriate to ask what an optimal dosage would be.

Unfortunately, the optimal dose of vitamin C is not established. Therecommended daily allowance sufficient to prevent scurvy varies from 30mg in the United Kingdom to 60 mg in the U.S. and 90 mg in the formerSoviet Union. In contrast, mega-doses of up to 16 g per day aresuggested to provide additional health benefits.

In fact, the optimal dose of vitamin C depends on many factors. It iswater soluble and cannot be stored in the body to any great extent.However, when vitamin C is regularly ingested, a body pool develops,which may become large even though some vitamin C is being excreted inthe urine. Thus, it can take months of ascorbic acid deprivation for thebody pool to become depleted to the point where symptoms of scurvyappear. Large-dose intake benefits may be small because of finiteabsorption from the intestine, limited metabolism by the liver, orexcretion by the kidney. For example, it has been reported that onlyabout 60% of a 500 mg dose is normally absorbed into the body of anaverage person within the first 12 hr after ingestion, while 40% isexcreted prior to metabolization (see Olson, J. A., and R. E. Hodges,"The Scientific Basis of the Suggested New RDA Values for Vitamins A andC," Nutr. Today, 20:14-15, 1985). In fact, it has been ascertained thata certain percentage of any dose is always excreted, and that suchpercentage increases with the dose (see Blanchard, J. et al., "Effectsof Age and Intake on Vitamin C Disposition in Females," Eur. J. Clin.Nutr., 44:447-460, 1990).

Without a clear physiological endpoint to measure an optimal dosage or acomplete understanding of the functional role of vitamin C, it isunclear how much vitamin C one should take. Because large doses are notentirely absorbed and may even irritate a patient's stomach, and becausesmall doses may be insufficient to provide sufficient anti-oxidantprotection, there exists a need to determine a dosage sufficiently largeto ensure as large as possible a presence in the body while at the sametime minimizing excesses and waste through excretion. This inventionprovides a method that fulfills this need by ensuring that some excessvitamin c is always present in the urine of a user.

BRIEF SUMMARY OF THE INVENTION

The primary objective of this invention is a dosage and regimen ofascorbic-acid intake that ensures continuously high levels thereof inthe body of a user without the need for large excesses at any giventime.

Another goal of the invention is an approach to determining optimalintake dosages and regimens for various kinds of water-soluble,urine-excreted, and non-toxic vitamins and other nutritional substances.

Therefore, according to these and other objectives, the presentinvention consists of a finding that a dose of about 500 mg takenapproximately every 12 hours produces a continuously-detectable level ofvitamin C in the urine of an average healthy person. If either one ofthese two conditions is not met, continuous excretion of vitamin C isnot maintained, thereby preventing the formation of a sufficiently highpool of ascorbic acid in the body to provide anti-oxidant protection.The minimum dosage and regimen found to be effective are respectivelysubstantially higher than the U.S. recommended daily allowance and morefrequent than administration rates previously used in clinical trials.

Various other purposes and advantages of the invention will become clearfrom its description in the specification that follows and from thenovel features particularly pointed out in the appended claims.Therefore, to the accomplishment of the objectives described above, thisinvention consists of the features hereinafter illustrated in thedrawings, fully described in the detailed description of the preferredembodiments and particularly pointed out in the claims. However, suchdrawings and description disclose only some of the various ways in whichthe invention may be practiced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the relationship between daily doses ofvitamin C and total urinary excretion during the 24-hour periodfollowing intake.

FIGS. 2A, 2B and 2C are an illustration of the rate of excretion ofvitamin C during beginning and end 24-hour periods of separate eight-daycycles of a single daily intake of two grams.

FIG. 3 illustrates the effect of two daily doses of vitamin C formaintaining a measurable excess in a taker's urine.

FIG. 4 shows the effect on excretion of vitamin C following a regimen oftwo daily doses of 500 mg each taken 12 hours apart.

FIG. 5 shows the effect of two daily doses of 500 mg of vitamin C takenfor two periods of eight consecutive days each.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention amounts to a finding of the minimum rate of intakeof ascorbic acid required to provide continuous optimal anti-oxidantprotection to an average individual. The invention also discloses ageneral method for determining such optimal dosages and intake regimensfor water-soluble vitamins and other nutritional supplements andmedicines that require elevated concentrations in the blood stream foroptimal effect.

Because vitamin C has been implicated in a variety of diseases, it hasbeen the subject of frequent clinical studies. These studies havecompared subjects taking vitamin C on a "regular daily basis" with thosenot taking vitamin C. However, because vitamin C is a water-solublevitamin, high doses are readily excreted in the urine, thereby quicklydepleting the system to blood concentrations well below saturation.

With reference to vitamin C, saturation is defined herein as the levelin an individual's blood stream above which vitamin C is excreted intothe urine. Thus, the work that resulted in this disclosure wasundertaken to determine what daily dose of vitamin C is necessary onaverage to produce a measurable level of vitamin C excretion in urine;whether a single dose, even a very high one, is sufficient to maintainmeasurable excretion at all times during an entire 24-hour period; andwhether taking a regular daily dose of vitamin C leads to any noticeablechange in vitamin C excretion over time.

The following procedures were followed in the experiments that generatedthe data shown in the figures.

I. Stability of Vitamin C in Water and Urine.

To determine whether vitamin C is stable in urine prior to assay, stocksolutions of vitamin C in water and in urine were prepared 48, 24, 12,and 0 hours before running standard curves using the DCIP procedurereferenced below. Each stock solution contained 125 μg of vitamin C/mlof 5% phosphoric acid in water or urine. The samples were stored in thedark at room temperature. The vitamin C degraded when stored in aqueoussolutions of 5% phosphoric acid, but was stable in solutions containingurine in 5% phosphoric acid.

II. Effect of Dose of Vitamin C on Total Urinary Excretion.

In order to determine how daily dosages related to total excretion andwhether high daily doses would alter the rate of excretion after a week,groups of test subjects took either 0, 0.25, 1, or 2 gram doses ofvitamin C at 8 am daily for 8 consecutive days. All urine was collectedand pooled on day 1 and on day 8. The concentration was assayed and thetotal amount of vitamin C excreted during the 24-hour period wasdetermined, as shown in FIG. 1. The figure shows that the rate ofurinary excretion of vitamin C increased with increasing daily doses ofvitamin C, and that the amount excreted was similar even after taking itfor 8 consecutive days.

III. Rate of Excretion of a Single Dose of Vitamin C.

In order to determine how rapidly vitamin C is excreted, severalindividuals took 2 grams of vitamin C daily (single dose) for 8consecutive days, and collected samples and measured the volume of everymicturition during the first and last day. The experiment was repeatedwith the same individuals after a seven-day rest. Each sample wasanalyzed to determine the time period, concentration range, and totalurinary excretion of vitamin C. The results are shown in FIG. 2 forthree representative individuals. Vitamin C concentration in the urineincreased after 4 hours from time of intake and remained elevated for atotal of 12 hours, consistent with the hypothesis that vitamin C levelsin the body are not elevated for an entire 24 hour period by a singledaily dose. However, only a small percentage of the ingested vitamin Cwas detected in the urine. No noticeable difference in the excretionpattern during the first and last days was observed.

IV. Effect of Twice-Daily Doses of Vitamin C on Urinary Output.

Because it was found that a single dose of vitamin C elevates urinelevels for only 12 hours and that a 500 mg dose is sufficient to providedetectable levels in the urine, one individual took doses from 250 to2000 mg of vitamin C every 12 hours to determine whether vitamin Clevels would remain continuously elevated. FIG. 3 shows that a dose of250 mg did not cause detectable excretion in amounts exceeding normalquantities, but doses of 500 mg or higher providedcontinuously-detectable elevated levels of excretion, with higher levelscorresponding to higher doses. Twice-daily doses of 500 mg weresufficient to elevate vitamin C in the urine continuously as shown for 5individuals in FIG. 4. When 500 mg are taken twice daily for a week,vitamin C remains continuously elevated on the first and last day, asshown for two such periods in FIG. 5.

Assay Procedures.

Vitamin C was assayed by the 2,6-dichlorophenolindophenol (DCIP) assay(see Omaye, T. S. et al. "Selected Methods for the Determination ofAscorbic Acid in Animal Cells," in Methods Enzymol.: Vitamins andCoenzymes, Vol. 62, edited by McCormick, D. B. and L. D. Wright, NewYork, Academic Press, 1979, pp. 3-11). Samples diluted to 0-40 μgascorbic acid with 5% phosphoric acid were assayed in a citrate/acetatebuffer by reaction with DCIP, and the color at 520 nm compared withstandards. Samples were assayed in duplicate and each experiment wasrepeated. The ascorbic acid standard was from Gibco Laboratories, andthe 500 mg vitamin C pills used were the brand marketed by Leiner HealthProducts, Inc., of Torrance, Calif., under the trademark "Your Life."The pills were assayed for vitamin C content by extraction by grindingin 5% phosphoric acid and were shown to contain the appropriate quantityof ascorbic acid.

We found that a one-time dose of 500 mg is necessary for detectableexcretion in most individuals. As shown in FIG. 1, doses of less than0.5 g per day caused no detectable vitamin C levels in urine, even whentaken for 8 consecutive days. Vitamin C was administered daily to fiveindividuals at 8 am for eight consecutive days. All urine for eachindividual was collected during the first and last 24-hour periods andthe total amount of vitamin C excreted in the urine was determined byDCIP assay. Doses of 500 mg to 2 g resulted in increased levels ofurinary vitamin C, so that it was readily detectable, but only forapproximately 12 hours even at the highest rate of intake. The figureshows that traces of ascorbic acid may be found in human urine at alltimes, even when no vitamin C has been administered in pills. Aconsistently elevated level (greater than the standard deviation of themeasurement SD) is found to begin at intake rates between approximately250 and 500 mg per day, thus showing that lower rates do not produce asaturated condition in the blood stream at all times.

FIG. 2 illustrates the effect of two grams of daily single-dose vitaminC taken orally for eight days, followed by six days with no intake, andthen by eight more days of two grams taken daily in a single dose. Eachmicturition during the first and last 24 hours of each eight-day periodwas collected and assayed to determine the total mass of vitamin Cexcreted. FIGS. 2a-2c represent excretion from three differentindividuals. Despite variations among the different subjects, FIG. 2shows that taking two grams daily for eight days did not lead to anynoticeable difference in the pattern of excretion from day to day forany of the subjects. This demonstrates that even a high single dailydose is inadequate to maintain an elevated rate of urinary excretion ofvitamin C on a continuous basis.

In order to achieve such sustained presence of vitamin C in urine, it isinstead necessary to take it at least twice daily, as shown in FIG. 3.Different dosages (from 0.25 to 2.0 grams) of vitamin C wereadministered to a representative subject every 12 hours, at times 0 and12 hour in the figure, on four separate days of a four week period. Themass of vitamin C present in the urine was measured at everymicturition. The individual was tested one day of the week for fourconsecutive weeks with progressively increasing dosages followed bysix-day periods of rest. As illustrated by the figure, taking at least500 mg twice daily led to continuously elevated levels of vitamin C inthe urine tested.

FIG. 4 shows the results on five individuals who took 500 mg of vitaminC at time 0 and 12 hour during a 24-hour period. Each micturition duringthe entire 24 hours was collected and the total mass of vitamin Cexcreted by each subject was measured by the DCIP assay. In yet anotherexperiment, two daily doses of 0.5 grams of vitamin C were taken dailyfor 8 days, then no pills were taken for a week, and then 0.5 g dailywere taken again for another 8 days. Each micturition during the firstand last 24 hr of each 8 day period was collected and assayed todetermine the total mass of vitamin C excreted. As seen in FIG. 5, thelevel of excretion was similar during the first and eighth day oftesting (demonstrating that no appreciable cumulative effect occurred).

These results have important implications for dietary purposes and forthe design of clinical studies involving vitamin C. First, small dosesof vitamin C are unlikely to provide elevated vitamin C levels in thebody, particularly in the blood. Therefore, optimal anti-oxidantprotection cannot be achieved below a minimum dosage threshold, found tobe at about 500 mg per dose. At this point it is still unclear whetherdoses below 500 mg/dose are absorbed or metabolized, but the evidenceclearly supports the fact that at least 500 mg/dose are necessary tosaturate the blood sufficiently for vitamin C to appear in the urine.

Second, although 500 mg/dose is sufficient to produce vitamin Cexcretion, a single dose of even 2 g/day is insufficient to maintainexcretion over a 24 hr period. Therefore, if a continuous protectiveeffect is desired or if a study is intended to determine particularprotective effects of vitamin C, it is necessary that the vitamin beadministered at least every 12 hours.

The precise role of vitamin C in maintaining good health remains to beunderstood, but a large body of evidence is consistent with thehypothesis that individuals taking doses of vitamin C substantiallygreater than the USDA Recommended Daily Allowance may have a substantialhealth benefit. Until the precise role of vitamin C becomes clear, itmay be prudent to take a dose which is large enough to producecontinuously detectable excretion, but not so large as to causedigestive-system discomfort or other problems. The results of thepresent work support the conclusion that in order to continuouslymaintain measurable levels of vitamin C in urine, at least 500 mg ofvitamin C should be taken at least twice daily (every 12 hours).

This work also demonstrated a useful tool to determine optimal dosagesand regimens for water soluble-substances that are desirable in elevatedconcentrations in the blood stream. By measuring the concentration ofsuch a substance in urine after ingestion of a predetermined dosage andaccording to a predetermined regimen, one can find an intake thresholdbelow which the substance is not maintained above saturation, therebysetting a minimum dosage and rate of administration for continuoussaturation in the blood pool of an individual. The dosage and regimenfor a variety of physiologically and medically useful substances clearlyvary for different individuals, depending on many personalcharacteristics such as age, weight, health, and gender. For substanceslike nutritional supplements, such as vitamins and minerals, andmedicines, such as aspirin, which are water-soluble, are excreted inurine, and are non-toxic at physiologically beneficial levels,monitoring detectable urinary excretion is a means to establish apotentially medically and/or nutritionally useful dosage and regimen.The method involves taking different dosages of substance at differentfrequencies and testing the urine to determine whether the substance ispresent in every micturition.

This method could also involve a variety of products specific to thesubstance being tested. For example, test strips containing individualpads of reactive materials which could be used to check a urine specimento determine whether a detectable quantity of a particular substance,such as vitamin C, salicylic acid, cystiene, zinc, or selenium, ispresent. Similarly, dropper kits would permit a person to add drops ofcertain substances to a urine specimen to determine whether a givensubstance is present in the urine at a detectable concentration.

For example, a kit for determining whether an individual is taking asufficient amount of vitamin C for a significant excess to be detectedin the urine could consist of the following components:

1. A dropper bottle, labelled Acid, containing a given quantity, such as10 ml, of 10% phosphoric acid, fitted with a dropper graduated at 0.6ml.

2. A dropper bottle, labelled Buffer, containing a commensuratequantity, such as 10 ml, of citrate/acetate buffer, pH 4.15 (made with2.2 g of trisodium citrate dihydrate), fitted with a dropper graduatedat 0.6 ml.

3. Two screw capped test tubes labelled 1 and 2 with graduations at 0.6ml, 1.2 ml, 1.8 ml, and 2.4 ml.

4. A dropper bottle at least 10 ml in capacity and containing 1 mg ofdry 2,6-dichlorophenolindophenol, labelled DCIP and fitted with adropper graduated at 0.6 ml.

5. A bottle containing a commensurate quantity, such as 10 ml, ofdistilled water, labelled Water.

6. A packet of vitamin C crystals (10 mg) labelled Vitamin C Crystals.

7. A plastic cup with a wide top, about 5 ml in volume, with a pour lipsimilar to beaker, labelled Collection Cup.

Such a kit is designed to permit a user to determine whether he or sheis getting enough vitamin C in the diet to be able to maintain acontinuous excretion of vitamin C in the urine. To be accurate, oneshould use this test throughout a single 24-hour period, testing asample at each time of urination, to see whether a sufficient amount ofvitamin C is kept in the system to be detected all day long. Becauseexcess vitamin C in the diet may be excreted by the kidneys within 12hours of reaching the bloodstream, it is important to monitor itspresence in the urine during an entire 24-hour period while consuming atypical diet. This kit is designed to allow one to do so safely andconveniently, using a single kit for an entire 24 hour period.

The kit is used as follows. At the start of the 24-hour period duringwhich one intends to test vitamin C excretion, the bottle of water isopened and emptied into the dropper bottle labelled DCIP, and thecombined contents are shaken for about 1 minute until all solids arecompletely dissolved, producing a blue DCIP solution.

A small sample of urine is collected in the Collection Cup each time theindividual urinates during the following 24 hours. Test tube 1 is filledup to the level of the first mark (0.6 ml) with the urine so collected.Then all further additions are made to test tube 1.

One dropper full (up to the 0.6-ml mark) of Acid is added to test tube1, which already contains the urine sample. Next, one dropper full(0.6-ml) of Buffer is added to test tube 1 (up to the 1.2-ml mark). Thecap is placed on test tube 1 and the contents are mixed by inverting itat least three times.

The contents of the test tube are then tested to check if there is anyvitamin C in the urine. Test tube 1 is opened and one dropper full (0.6ml) of DCIP solution is added (up to the 1.8-ml mark). If the bluishcolor of the DCIP solution immediately disappears, vitamin C is presentin the urine. If instead a pink color appears and persists for more than15 seconds, the cap is replaced and the tube inverted several times formixing. If the pink color now disappears, it is an indication that somevitamin C is excreted in the urine. If the pink color still persists, orif the color cannot be seen clearly because of the color of the urineitself, the procedure is continued as follows.

Half the contents of test tube 1 are poured into test tube 2, so the twosolutions are divided approximately evenly between the two tubes. Thepacket of Vitamin C Crystals is opened and a few crystals (one to three)are sprinkled into test tube 1. The tube is capped and its contentsmixed by inverting it at least three times.

The colors of tube 1 and tube 2 are now compared. If the two tubes areidentical in color, it is an indication that a sufficient amount ofvitamin C is being consumed and that the urine sample being testedcontains excess vitamin C. If tube 2 is darker or more strongly pinkthan tube 1, then it is an indication that no excess is present and thatpossibly one would benefit from consuming additional vitamin C through abetter diet or vitamin supplements.

This test is repeated at each micturition during the chosen 24-hourperiod. Because vitamin C is excreted rapidly from the body, vitamin Cis unlikely to be present in urine more than 16 hours after a goodsource of vitamin C is last consumed. Therefore, one should check for anentire 24-hour period to determine if enough vitamin C is being consumedon a regular basis to keep a continuously-elevated level in urine.

Note that the kit and procedure just described represent a simple methodfor implementing the concept of this invention. However, many similarstrategies and devices could be used to achieve the same end. Forinstance, the reagents could be attached in powder form to a stiff,insoluble, plastic stick, such that all of the reagents except the dyewould dissolve off of the stick when stirred in a small cup of urine,and then the test stick removed, and the color of the stick wouldindicate the presence or absence of vitamin C by the change in thedye-containing region of the stick. Note also that certain substancespresent in the urine of some individuals may interfere with the resultsof the test. For instance, consumption of beets will turn urinetemporarily red, which will make it difficult to observe any resultsclearly.

A person could use this method, implemented through a product asdescribed above, as a simple indication of whether a sufficient dosageand regimen of a substance were being taken to ensure the presence of aminimum useful excess of the desired substance in the body at all times.The method could similarly be used by researchers to gather data for avariety of substances by age, weight, and other criteria, to establish adosage which would ensure the presence of a minimum useful excess of asubstance in the body. As is known for vitamin C, 60 mg daily willprevent scurvy, but much higher doses may be required to improve health,such as by preventing a variety of adult disorders not readily connectedwith a lack of vitamin C. By application of the method of the invention,500 mg of vitamin C twice daily has been determined to provide asufficient dose to have excess vitamin C excreted. The method maysimilarly be applied to determine a useful level of aspirin as a bloodthinner to prevent heart disease, and for other water-soluble vitaminsand minerals such as cystiene, zinc, and selenium, which may beantioxidants, for calcium, and for many other useful substances fittingthe three criteria of water solubility, excretion in urine, andnon-toxicity at physiologically beneficial levels.

Various other changes in the details, steps and substances that havebeen described may be made by those skilled in the art within theprinciples and scope of the invention herein illustrated and defined inthe appended claims. Thus, while the present invention has been shownand described herein in what is believed to be the most practical andpreferred embodiment, it is recognized that departures can be madetherefrom within the scope of the invention, which is not to be limitedto the details disclosed herein but is to be accorded the full scope ofthe claims so as to embrace any and all equivalent apparatus andmethods.

I claim:
 1. A test kit for determining the optimal level of intake foran individual of a useful substance that is water soluble, urineexcretable, and nontoxic at physiologically beneficial levels,consisting essentially of:container means for holding a sample of urinefrom said individual; reagent means for reacting with an amount of saidsubstance contained in a sample of urine from said individual; andvisual means for indicating to a user a concentration of said substancein said sample of urine in response to a reaction between said reagentmeans and said amount of said substance.
 2. The test kit of claim 1wherein said substance is vitamin C.
 3. The test kit of claim 2 whereinsaid visual means consists of 2,6-dichlorophenolindophenol dissolved inwater.
 4. The test kit of claim 2 wherein said reagent means consists ofphosphoric acid, a buffer of trisodium citrate dehydrate, and vitamin C.5. The test kit of claim 4 wherein said visual means consists of2,6-dichlorophenolindophenol dissolved in water.
 6. A test kit fordetermining the optimal level of intake for an individual of vitamin C,consisting essentially of:container means for holding a sample of urinefrom said individual; reagent means for reacting with an amount of saidvitamin C contained in said sample of urine from said individual; saidreagent means consisting of phosphoric acid, a buffer of trisodiumcitrate dihydrate, and vitamin C; visual means for indicating to a usera concentration of said vitamin C in said sample of urine in response toa reaction between said reagent means and said amount of said substance;and said visual means consisting of 2,6-dichlorophenolindophenoldissolved in water.
 7. A kit for determining Vitamin C depletion in anindividual urine donor, consisting essentially of:phosphoric acid; abuffer of trisodium citrate dehydrate; 2, 6-dichlorophenolindophenol(DCIP) dissolved in water; a first container for mixing the phosphoricacid, buffer and (DCIP) with urine from the donor, wherein the presenceof color in the container when the DCIP is combined with the urine, acidand buffer indicates the urine donor is Vitamin C depleted; Vitamin C;and a second container for combining the Vitamin C with a portion of theDCIP, urine, acid and buffer combination wherein the same color in thefirst and second containers indicates no Vitamin C depletion but adarker color in the first container as compared to the second containerconfirms Vitamin C depletion.
 8. The kit of claim 7 wherein thephosphoric acid comprises 10% phosphoric acid.
 9. The kit of claim 7wherein the amount of buffer comprises and amount commensurate inquantity to the phosphoric acid, the buffer made with 2.2 g of trisodiumcitrate dihydrate with a pH of 4.15.
 10. The kit of claim 7 wherein theamount of DCIP comprises an amount commensurate in quantity to thephosphoric acid and buffer.